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2Department of Biology, Fukuoka University of Education, 729-1 Akama, Munakata, 811-41 Japan; and 3Department of Biology, Faculty of Education, Gifu University, Gifu, 501-11 Japan
Received for publication October 28, 1997. Accepted for publication June 16, 1998.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: allozyme • dichogamy • genetic diversity • geographic range • Liliaceae • population genetic structure • Tricyrtis sect. Flavae
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Many factors are known to affect genetic diversity and the population genetic structure of a plant species (Loveless and Hamrick, 1984
). One of these factors is the geographic range of a species. Widespread species tend to have more genetic variability than close relatives with a narrow distribution (Karron, 1987
; Hamrick and Godt, 1990
), although geographic range appears to have little influence on the interpopulational differentiation of the species (Hamrick and Godt, 1990
) Another factor affecting genetic diversity and genetic partitioning is the mating system of a species. Selfing species are prone to have a lower level of intrapopulation genetic diversity than outcrossing species, and in a selfing species each population tends to be more differentiated from the others (Hamrick and Godt, 1990
). This is probably because gene flow via pollen is more limited in selfing species than in outcrossing congeners.
Tricyrtis sect. Flavae (Liliaceae) consists of four species: T. flava Maxim., T. nana Yatabe, T. ohsumiensis Masamune, and T. perfoliata Masamune. Among them, only T. nana is adichogamous, and other species are protandrous (Takahashi, 1987a
, 1998
). Tricyrtis nana usually have one or two flowers, whereas the others often have more than ten flowers (Satake, 1982
). These facts suggest that T. nana is a selfing species, while the others mainly outcross (Ornduff, 1969
).
Tricyrtis nana has the widest distribution among the four species; T. flava is restricted to a relatively narrow area, and T. ohsumiensis and T. perfoliata are endemic to a few mountains in the Ohsumi Peninsula and Mt. Osuzu, respectively.
In this study, we employed enzyme electrophoresis to examine genetic diversity and the population genetic structure in Tricyrtis sect. Flavae. In particular, we investigated the effect of geographic range and dichogamy on genetic variability and genetic partitioning among populations of the species. In addition, we discuss genetic similarity among the four species based on the genetic distance estimated from allozyme frequencies.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). The chromosome numbers of the species are 2n = 26, indicating that the species are diploid (Takahashi, 1980
). Tricyrtis nana and T. flava are usually found on roadsides and the understory or edge of evergreen forests, whereas T. ohsumiensis and T. perfoliata are found on sunny rocks and wet cliffs under evergreen forests, respectively. Tricyrtis nana is distributed in the central part of Honshu, the southern part of Kii Peninsula, Shikoku Island, and Kyusyu Island of Japan (Takahashi, 1987b
, unpublished data). Tricyrtis flava is restricted to the southeastern part of Kyusyu Island, and T. perfoliata and T. ohsumiensis are endemic to Mt. Osuzu and a few mountains in the Ohsumi Peninsula, respectively (Takahashi, 1987b
). The plant red list of Japan, which adopted the new Red List Categories proposed by IUCN (1994)
, names T. perfoliata, T. flava, and T. ohsumiensis as endangered, vulnerable, and near-threatened species, respectively (Anonymous, 1997
). Individual flowers of T. nana open for 1 d in late summer or early autumn, and anther dehiscence and stigma maturity occur simultaneously, while individual flowers of T. flava and T. ohsumiensis open for 2 d in late autumn, and the flowers are male-stage on the first day and female-stage on the second day (Takahashi, 1987a
). Although the flowering pattern of T. perfoliata is similar to T. flava and T. ohsumiensis, its flowering period lasts for 4–5 d. The dominant pollinator of all four species is Bombus diversus (Takahashi, 1987a
, 1998
). Bagging experiments showed that T. nana, T. ohsumiensis, and T. flava are self-compatible (Takahashi, 1987a
). It is not certain whether T. perfoliata is self-compatible or not.
Owing to the morphological resemblance, small individuals of T. flava have often been mistaken for T. nana (e.g., Hayashi, Azegami, and Hishiyama, 1983
). A phylogenetic study using restriction fragment polymorphism of chloroplast DNA revealed that T. nana is more closely related to T. ohsumiensis than to T. flava and that T. perfoliata and the other three Flavae species do not cluster together, suggesting that sect. Flavae is polyphyletic (Kato et al., unpublished data).
Sampling populations
We sampled five and seven populations of T. flava and T. nana, respectively, to represent the whole geographic range of the species, and one population each of T. ohsumiensis and T. perfoliata because few populations large enough for population genetic study exist in these two species. The population code and the number of samples are given in Fig. 1. We collected a portion of mature leaves individually from the populations and then transported them on ice to the laboratory. The samples were kept in a refrigerator for up to 2 wk until electrophoresis was carried out. At 2 wk after sampling, enzyme activity did not decrease.
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). The homogenates were centrifuged at 15 000 rpm at 4° C for 30 min. The resultant supernatant was used for crude extract of the enzymes.
Polyacrylamide and starch gel electrophoresis were employed. Polyacrylamide vertical slab gel electrophoresis was carried out according to the procedures described by Davis (1964)
and Ornstein (1964)
. Ten microlitres of the crude extract of enzyme were used for electrophoresis for each enzyme for polyacrylamide gel electrophoresis. The following 13 enzyme systems were examined by polyacrylamide gel electrophoresis: asparate amino transferase (AAT; EC 2.6.1.1), colometric esterase (EST; EC 3.1.1), formate dehydrogenase (FDH; EC 1.2.1.2), fructose-bisphosphatase (FBP; EC 3.1.3.11), glucose-6-dehydrogenase (G6PDH; EC 1.1.1.49), glucose-6-phosphate isomerase (GPI; EC 5.3.1.9), glutamate dehydrogenase (GDH; EC 2.7.1.1), malate dehydrogenase (MDH; EC 1.1.1.40), phosphoglucomutase (PGM; EC 5.4.2.2), 6-phosphogluconate dehydrogenase (6PGDH; EC 1.1.1.44), shikimate dehydrogenase(SKDH; EC 1.1.1.25), superoxide dismutase (SOD; 1.15.1.1), and triose-phosphate isomerase (TPI; EC 5.3.1.1). For starch gel electrophoresis, the enzyme crude extract was absorbed onto a 4 x 14 mm wick cut from Whatman 3MM chromatography paper. Electrode and gel buffer system 2 of Soltis et al. (1983)
was used to resolve the following two enzyme species: malic enzyme (ME; EC 1.1.1.40), [NADP] glycelaldehyde-3-phosphate dehydrogenase ([NADP] G3PDH; EC 1.2.1.12). Staining protocols followed Tsumura et al. (1990), except FBP, [NADP]G3PDH, and FDH, which followed Soltis et al. (1983)
, Rieseberg et al. (1987)
, and Wendel and Weeden (1989)
, respectively. SOD was scored on both TPI and MDH gels.
Statistical analysis
Allele frequencies in each population of the species were calculated for the loci encoding the 15 enzyme systems. The following indices were used to quantify the amount of genetic diversity within each population of the four species examined: the proportion of polymorphic loci (P) at 95% criterion, the number of alleles per polymorphic loci (AP), the number of alleles per locus (A) , and the expected heterozygosity (h).
Genetic differentiation among the populations of T. nana and T. flava was estimated by Nei's gene diversity statistics (Nei, 1973
) for polymorphic loci: total genetic diversity (HT), genetic diversity within populations (HS), and proportion of the total diversity among populations (GST). The amount of gene flow among these populations was estimated as Nm = (1/GST-1)/4 (Slatkin and Barton, 1989
).
Genetic diversity parameters (P, A, AP, and h) were also calculated at the species level for T. nana and T. flava. As in Hamrick and Godt (1990)
, we treated the loci polymorphic in at least one population as polymorphic at the species level.
Genetic identity and standard genetic distance (Nei, 1972
) for each pairwise comparison of all populations examined were calculated. We obtained a phenogram based on the standard genetic distance using the neighbor joining method (Saitou and Nei, 1987
).
Wright's (1951)
fixation index (f) was estimated at each polymorphic locus as f = 1 - no/ne, where no is the observed number of heterozygotes, and ne is the Hardy-Weinberg expected number of heterozygotes. For loci with more than two alleles, the frequencies of the less frequent alleles were combined into a single class. Chi-square values for each locus in a population were calculated as Nf 2, where N is the number of individuals per population (Li and Horvitz, 1951
).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Genetic diversity at the population level of T. flava is comparable to that of the species with narrow geographic range.
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). Although T. flava had several polymorphic loci at the species level, the mean number of alleles (A) was lower than and the expected heterozygosity (h) was comparable to endemic species.
Population genetic structure
Table 2 summarizes the gene diversity statistics based on Nei (1973)
. The levels of genetic differentiation among populations were relatively large in both T. flava and T. nana. The GST value for T. nana was comparable to and the value for T. flava was slightly lower than selfing species (GST = 0.510) (Hamrick and Godt, 1990
). Gene flow per generation is highly limited in both T. flava (Nm = 0.36) and T. nana (Nm = 0.27).
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). Figure 2 is a phenogram constructed using the neighbor-joining method based on Nei's (1972)
standard genetic distance. Tricyrtis nana appears to be recently derived from an ancestral population of T. flava.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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; Hamrick and Godt, 1990
). Many studies have revealed that narrowly restricted species tend to have a lower level of genetic diversity than the widespread congeners (Karron, 1987
; Sherman-Broyles et al., 1992
; Linhart and Premoli, 1993
; Edwards and Wyatt, 1994
, Purdy and Bayer, 1995a
, b
, 1996
; Godt, Walker, and Hamrick, 1997
). A few studies, however, have reported the opposite pattern (Ranker, 1994
; Lewis and Crawford, 1995
). In 11 North American species of Polygonella, the two most widespread species have reduced gene diversity with respect to their narrowly endemic congeners (Lewis and Crawford, 1995
). These unexpected results were explained as being due to the high levels of self-fertilization for the widespread species and a large-scale migration during Pleistocene glaciation. As in North American Polygonella, T. nana, the most widely spread species, has the lowest level of genetic diversity, and the narrowly restricted species T. flava and two endemic species T. perfoliata and T. ohsumiensis have a relatively high genetic variability.
For intrapopulation genetic diversity, the mating system plays a major role in determining the level of genetic diversity; selfing species tend to maintain a lower proportion of their genetic variability within a population than do outcrossing species (Hamrick and Godt, 1990
). This is also the case for Tricyrtis sect. Flavae species; T. nana is an adichogamous and putative selfing species, whereas the remaining three species are typically protandrous and putative outcrossing species.
On the other hand, at the species level, differences in the level of genetic diversity in outcrossing and selfing species are not obvious (Hamrick and Godt, 1990
). The depauperate genetic diversity at the species level of T. nana is probably a result of a bottleneck effect at speciation. Inferring from the phenogram constructed in this study (Fig. 2), T. nana was derived from T. flava or its ancestor relatively recently, and it has rapidly enlarged its area of distribution. The selfing nature of T. nana may have played a major role in this quick expansion. Although other explanations such as the migration during Pleistocene glaciation as in North American Polygonella are possible, there is no evidence supporting other hypotheses.
Population genetic structure
Population genetic structure is more differentiated in selfing species than in outcrossing species. In the putative selfing species T. nana, it is reasonable to assume that the population genetic structure is differentiated and that interpopulation gene flow is highly limited, probably because gene flow via pollen is extremely restricted owing to the selfing nature of the species. The putative outcrossing species T. flava, however, also has a differentiated population genetic structure, and this is an unexpected finding. One possible explanation is that T. flava is a colonizing species. In species that repeat the extinction and colonization of populations, genetic differentiation among populations tends to be inflated compared to species that do not have metapopulation dynamics (Whitlock and McCauley, 1990
; McCauley, Raveil, and Antonovics, 1995
). Because T. flava commonly grows at the roadside or the edge of the forest where the environment is frequently disturbed, its population turnover appears to be rapid, leading to a differentiated population genetic structure. The colonizing habit may also contribute to interpopulation genetic differentiation in T. nana, which grows on roadsides and forest edges
Genetic similarities among the species
The previous phylogenetic study for Tricyrtis sect. Flavae using the RFLP (restriction fragment length polymorphism) of chloroplast DNA revealed that T. nana is most closely related to T. ohsumiensis. However, the present study showed that T. nana is more closely related to T. flava than to T. ohsumiensis, which corresponds to their morphological resemblance. In addition, the genetic distances between T. ohsumiensis and other species are relatively large (Table 5). Some studies similarly found a difference between phylogenies based on chloroplast DNA (cpDNA) and on allozymes (references in Rieseberg and Soltis, 1991
). At present, it is not clear what causes this discrepancy between the allozyme and the cpDNA studies in sect. Flavae. One explanation might be the sampling error of the cpDNA study. Only one site change was found in T. flava and the two species (T. nana and T. ohsumiensis). Another explanation might be chloroplast capture through past introgressive hybridization. Much evidence for chloroplast capture has been obtained for wild plants (Rieseberg and Soltis, 1991
; Rieseberg and Wendel, 1993
). Chloroplast DNA of T. ohsumiensis may have been derived from T. nana by chloroplast capture. More resolvable estimates using the sequence variations of cpDNA, together with nuclear DNA variation such as ITS region, are necessary for the elucidation of the relationships between T. nana and the other related species.
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FOOTNOTES |
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5 Current address: Department of Systems Science (Biology), University of Tokyo, Komaba, Meguro-ku, Tokyo, 153-8902 Japan.
6 Current address: Department of Ecology and Evolutionary Biology, Graduate School of Science, Tohoku University, Aoba, Sendai, 980-8578 Japan.
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LITERATURE CITED |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Crawford, D. J. 1990 Plant molecular systematics. Wiley-Interscience, New York, NY.
Davis, B. J. 1964 Disk electrophoresis. II. Method and application to human serum proteins. Annals of the New York Academy of Sciences 121: 404–427.
Edwards, A. L., and R. Wyatt. 1994 Population genetics of the rare Asclepias texana and its widespread sister species, A. perennis. Systematic Botany 19: 291–307.
Futuyma, D. J. 1986 Evolutionary biology, 2d ed. Sinauer, Sunderland, MA.
Godt, M. J. W., J. Walker, and J. L. Hamrick. 1997 Genetic diversity in the endangered lily Harperocallis flava and a close relative, Tofieldia racemosa. Conservation Biology 11: 361–366.
Hamrick, J. L., and M. J. Godt. 1990 Allozyme diversity in plant species. In A. H. D. Brown, M. T. Clegg, A. L. Kahler, and B. S. Weir [eds.], Plant population genetics, breeding, and genetic resources, 43–63. Sinauer, Sunderland, MA.
Hayashi, Y., T. Azegami, and T. Hishiyama. 1983 Wild flowers of Japan. Yama-to-Keikoku-sya, Tokyo, Japan (in Japanese).
IUCN (International Union for Conservation of Nature). 1994 IUCN Red List Categories. IUCN, Switzerland.
Karron, J. D. 1987 A comparison of levels of genetic polymorphism and self-incompatibility in geographically restricted and widespread plant congeners. Evolutionary Ecology 1: 47–58.
Lewis, P. O., and D. J. Crawford. 1995 Pleistocene refugium endemics exhibit greater allozyme diversity than widespread congeners in the genus Polygonella (Polygonaceae). American Journal of Botany 82: 141–149.
Li, C. C., and D. G. Horvitz. 1951 Some methods of estimating the inbreeding coefficient. American Journal of Human Genetics 5: 107–117.[ISI]
Linhart, Y. B., and A. C. Premoli. 1993 Genetic variation in Altes acaulis and its relative, the narrow endemic A. humilis (Apiaceae). American Journal of Botany 80: 598–605.
Loveless, M. D., and J. L. Hamrick. 1984 Ecological determinants of genetic structure in plant populations. Annual Review of Ecology and Systematics 15: 65–96.
McCauley, D. E., J. Raveil, and J. Antonovics. 1995 Local founding events as determinants of genetic structure in a plant metapopulation. Heredity 75: 630–636.[ISI]
Nei, M. 1972 Genetic distance between populations. American Naturalist 106: 283–292.[CrossRef][ISI]
———. 1973 Analysis of gene diversity in subdivided populations. Proceedings of the National Academy of Sciences, USA 70: 3321–3323.
Ornduff, R. 1969 Reproductive biology in relation to systematics. Taxon 18: 121–133.[CrossRef]
Ornstein, N. L. 1964 Disk electrophoresis. I. Background and theory. Annals of the New York Academy of Sciences 121: 321–349.
Purdy, B. G., and R. J. Bayer. 1995a Allozyme variation in the Athabasca sand dune endemic, Salix silicicola, and the closely related widespread species, S. alaxensis. Systematic Botany 20: 179–190.
———, and ———. 1995b Genetic diversity in the tetraploid sand dune endemic Deschampia mackenzieana and its widespread progenitor D. cespitosa (Poaceae). American Journal of Botany 82: 121–130.[CrossRef][ISI]
———, and ———. 1996 Genetic variation in populations of the endemic Achillea millefolium ssp. megacephala from the Athabasca sand dunes and the widespread ssp. lanulosa in western North America. Canadian Journal of Botany 74: 1138–1146.
Ranker, T. A. 1994 Evolution of high genetic variability in the rare Hawaiian fern Adenophorus periens and implications for conservation management. Biological Conservation 70: 19–24.
Rieseberg, L. H., P. M. Peterson, D. E. Soltis, and C. R. Annable. 1987 Genetic divergence and isozyme number variation among four varieties of Allium douglasii (Alliaceae). American Journal of Botany 74: 1614–1624.[CrossRef][ISI]
———, and D. E. Soltis. 1991 Phylogenetic consequences of cytoplasmic gene flow in plants. Evolutionary Trends in Plants 5: 65–84.[ISI]
———, and J. F. Wendel. 1993 Introgression and its consequences in plants. In R. G. Harrison [ed.], Hybrid zones and the evolutionary process, 70–109. Oxford University Press, New York, NY.
Saitou, N., and M. Nei. 1987 The neighbor joining method: a new method for reconstructing phylogenetic trees. Molecular Biology and Evolution 4: 406–425.[Abstract]
Satake, Y. 1982 Liliaceae. In Y. Satake, J. Ohwi, S. Kitamura, S. Watari, and T. Tominari [eds.], Wild flowers of Japan, vol. 1, 21–51. Kodansha, Tokyo, Japan (in Japanese).
Sherman-Broyles, S. L., J. P. Gibson, J. L. Hamrick, M. A. Bucher, and M. J. Gibson. 1992 Comparison of allozyme diversity among rare and widespread Rhus species. Systematic Botany 17: 551–559.
Slatkin, M., and N. H. Barton. 1989 A comparison of three indirect methods for estimating average levels of gene flow. Evolution 43: 1349–1368.[CrossRef][ISI]
Soltis, D. E., C. H. Haufler, D. C. Darrow, and G. J. G. 1983 Starch gel electrophoresis of ferns: a compilation of grinding buffers, gel and electrode buffer, and staining schedules. American Fern Journal 73: 9–27.[CrossRef][ISI]
Takahashi, H. 1980 A taxonomic study on the genus Tricyrtis. Science Reports of the Faculty of Education, Gifu University 6: 583–635.
———. 1987a A comparative floral and pollination biology of Tricyrtis flava Maxim., T. nana Yatabe and T. ohsumiensis Masamune (Liliaceae). Botanical Magazine, Tokyo 100: 185–203.
———. 1987b Distribution of Tricyrtis and its phytogeographical problems. Acta Phytotaxonomica et Geobotanica 38: 123–132 (in Japanese).
———. 1998 Pollination biology of Tricyrtis perfoliata Masamune (Liliaceae). Memoirs of the National Science Museum 30: 57–63.
Uchida, K., Y. Tsumura, and K. Ohba. 1991 Inheritance of isozyme variants in leaf tissues of Hinoki, Chamaecyparis obtusa, and allozyme diversity of two natural forests. Japanese Journal of Breeding 41: 11–24.
Wendel, J. F., and N. F. Weeden. 1989 Visualization and interpretation of plant isozymes. In D. E. Soltis and P. S. Soltis [eds.], Isozymes in plant biology, 5–45. Dioscorides Press, Portland, OR.
Whitlock, M. C., and D. E. McCauley. 1990 Some population genetic consequences of colony formation and extinction: genetic correlations within founding groups. Evolution 44: 1717–1724.[CrossRef][ISI]
Wright, S. 1951 The genetical structure of populations. Annals of Eugenics 15: 323–354.[ISI]
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= T. flava. 
= T. perfoliata. 
= T. ohsumiensis.